Operational Qualification and

Performance Verification of
UV-visible Spectrophotometers

Technical Note

In principle, operational qualifica-

tion and performance verification
(OQ/PV) is not difficult to per-
form, because spectrophotome-
ters are relatively simple analyti-
cal instruments. However, in prac-
tice there are significant problems
because of the need to conform
closely to regulatory requirements
and because of the non-availability
of appropriate and easy-to-use
standards.

This technical note reviews the

regulatory requirements, the
advantages and disadvantages of
the standards currently available
and describes a new approach
which significantly reduces the
time required to perform OQ/PV.

Introduction mendations of pharmacopeias

In recent years quality require-
ments, as outlined by ISO 9000,
GLP, GMP and NAMAS, have
assumed increasing importance
and as a consequence, in the phar-
maceutical industry, the recom-
have also become more influen-
tial. Verification of the continued
proper performance of UV-visible
spectrophotometers is an impor-
tant element of these quality
requirements.
Definition
The terms operational qualification and perfor-
mance verification are used interchangeably in
this publication. Both terms are used to mean a
series of tests designed to prove that an instru-
ment is working within a set of required specifi-
cations.

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Regulatory requirements
There are many regulatory
requirements that govern the use
of UV-visible spectrophotometers.
The following sections review the
most important of these.
Good laboratory practice and
good manufacturing practice
Good laboratory practice (GLP)
and good manufacturing practice
(GMP) requirements concerning
the validation of instruments can
be summarized as:
“Documented verification that
the system or subsystem per-
forms as intended throughout
representative or anticipated
operating ranges.” 1
In reference to spectroscopy, the
following is suggested:
“Where appropriate, periodic
performance checks should be
carried out (for example, …
the resolution, alignment and
wavelength accuracy of spec-
trophotometers, etc.).” 2
The statement intended and It is worthwhile noting that the
anticipated operating ranges is American Standard Testing
critical when considering an Methods (ASTM) and the National
OQ/PV strategy. For example, if Institute of Standards and
the intended purpose is to mea- Technology (NIST) make similar
sure absorbance in the UV region statements. For example, the
(as is the case for most pharma- ASTM states that the wavelengths
ceutical analyses), it is not appro- used for calibration should, if pos-
priate to verify photometric accu- sible, bracket the analytical wave-
racy in the visible range—it must length and NIST comments on the
be done in the UV region and use of their various standards.
preferably at several wavelengths.
Similarly, it is not appropriate to
verify wavelength accuracy at the
656.1 nm deuterium line because
this is not a reliable indicator of
wavelength accuracy in the UV
region. Further, as the vast majori-
ty of samples to be measured in
the pharmaceutical laboratory are
solutions contained in cuvettes, it
can be argued that the use of solid
or emission standards is inappro-
priate, because these standards
have different optical characteris-
tics from the samples that will be
analyzed.
United States Pharmacopeia3
The regulatory requirements for
UV-visible spectrophotometers are
defined in the United States
Pharmacopeia (USP) XIII, Section
851 on “Spectrophotometry and
Light Scattering.” It states:
“Check the instrument for
accuracy of calibration. … The
wavelength scale may be cali-
brated also by means of suit-
able glass filters, which have
useful absorption bands
through the visible and ultravi-
olet regions. Standard glasses
containing didymium (a mix-
ture of praseodymium and
neodymium) have been widely
used. Glass containing holmi-
um is considered superior.
For checking the photometric
scale, a number of standard
inorganic glass filters as well
as standard solutions of
known transmittance such as
potassium chromate or potas-
sium dichromate are avail-
able.”
The latter contains a cross-refer- The NIST4 provides a range of
ence: solid and liquid standards for
determining wavelength accuracy,
“For further details regarding photometric accuracy and stray
checks on both wavelength and light. The most important are sum-
photometric scales of a spec- marized in table 1.
trophotometer, reference may
be made to the following publi-
cations of the National
Institute of Science and
Technology …”
SRM# Type NIST Description
930 Neutral density glass filters This SRM is for the verification and calibration
of the transmittance and absorbance scales of
visible absorption spectrometers.
931 Cobalt and nickel solution in This SRM is for the verification and calibration
nitric/perchloric acid mixture of the absorbance scales of ultraviolet and visible
absorption spectrometers having narrow band
passes.
935 Potassium dichromate solid for This SRM is for the verification and calibration
preparation of test solution of the transmittance and absorbance scales of
ultraviolet absorption spectrometers.
2031 Metal-on-quartz This SRM is for the verification and calibration
of the transmittance and absorbance scales of
ultraviolet and visible absorption spectrometers.
2034 Holmium oxide solution in This SRM is for use in the verification and
perchloric acid calibration of the wavelength scale of ultraviolet
and visible absorption spectrometers having
nominal spectral bandwidths not exceeding 3 nm.
2032 Potassium iodide solid for This SRM is for use in the assessment of
preparation of test solution heterochromic stray radiant energy (stray light) in
ultraviolet absorption spectrophotometers.
Table 1
NIST standards
European Pharmacopoeia5
Control of wavelengths—Verify the wavelength scale using the
The European Pharmacopoeia absorption maxima of holmium perchlorate solution R, the line of
(EP) is based on the requirements a hydrogen or deuterium discharge lamp or the lines of a mercury
of national pharmacopoeias such vapor arc shown below. The permitted tolerance is ± 1 nm for the
as the British Pharmacopoeia (BP) UV range and ± 3 nm for the visible range.
and the Deutsche Arzneimittel-
buch (DAB) in Germany, and is 241.15 nm (Ho) 404.66 nm (Hg)
253.7 nm (Hg) 435.83 nm (Hg)
very specific in its requirements 287.15 nm (Ho) 486.0 nm (Db)
for UV-visible spectrophotome- 302.25 nm (Hg) 486.1 nm (Hb)
ters. The EP requirements are 313.16 nm (Hg) 536.3 nm (Ho)
334.15 nm (Hg) 546.07 nm (Hg)
described in the following sec- 361.5 nm (Ho) 576.96 nm (Hg)
tions. 365.48 nm (Hg) 579.07 nm (Hg)

Note that the British
Pharmacopoeia states that the
ratio should “… be not less than
1.5 unless otherwise specified in
the monograph” and this is often
taken as a de facto minimum per-
formance specification for phar-
maceutical applications.
Control of absorbance—Check the absorbance using a solution of
potassium dichromate R at the wavelengths indicated in the follow-
ing table, which gives for each wavelength the exact values of the
specific absorbance and the permitted limits. The tolerance for the
absorbance is ± 0.001.
Wavelength (nm) A (1 percent, 1 cm) Maximum Tolerance

235 124.5 122.9 to 125.2

257 144.0 142.4 to 145.7
313 48.6 47.0 to 50.3
350 106.6 104.9 to 108.2

Limit of stray light—Stray light may be detected at a given wave-

length with suitable filters or solutions. For example the
absorbance of a 1.2 percent m/V solution of potassium chloride R
in a 1-cm cell should be greater than two at 200 nm when com-
pared with water R as compensation liquid.

Resolution power—When prescribed in a monograph, measure

the resolution of the apparatus as follows: record the spectrum of a
0.02 % V/V solution of toluene R in hexane R. The minimum ratio
of the absorbance at the maximum at 269 nm to that at the mini-
mum at 266 nm is stated in the monograph.
Recommended OQ/PV test
parameters and standards
When the above regulatory
requirements are considered
together there are many inconsis-
tencies and, in some cases, dis-
agreements. However, it is possi-
ble to define a strategy of tests
and standards that essentially sat-
isfies all requirements. This is
illustrated in table 2 where the
implied USP methods (based on
NIST standards) and the EP meth-
ods are summarized and then the
GLP filter of suitability for pur-
pose (liquid standards calibrated
in the UV range) is laid over the
matrix.
In addition, although not specified
by the USP or EP, it is generally
accepted that to characterize
properly the performance of a
spectrophotometer the parameters
noise, baseline flatness and stabili-
ty should also be tested.
Linearity is also often considered
to be an important factor for per-
formance verification. However,
the primary instrumental reason
for non-linearity is tested for.
Other reasons for non-linearity are
strongly sample or sampling
dependent and for this reason it is
the authors opinion that this para-
meter is best measured during a
system suitability test as
described below.
Type of Test USP EP Comment
Wavelength accuracy
• Deuterium discharge lamp 4 Only two peaks
• Mercury discharge lamp 4
• Holmium oxide glass 4 Position of peaks may vary from batch to batch
and must be calibrated.
• Holmium perchlorate solution 4 4 Peaks throughout UV and visible ranges.
Photometric accuracy
• Neutral density glass filters 4 Blocks in UV range so only useable for visible
range
• Metal on quartz filters 4 Often problems with interreflection errors and
very temperature sensitive
• Potassium dichromate solution 4 4 Has a tendency to decompose
Stray light
• Sodium nitrite solution Not essential but gives a good data point in the
mid-UV range at 340 nm
• Potassium iodide solution 4 Use for 220 nm
• Potassium chloride solution 4 Use for 200 nm
Resolution
• Toluene in Hexane 4
Table 2
Regulatory test requirements
The major problem in doing per- the performance characteristics
formance verification is the fact have changed in a way which
that all of the above are wave- could affect the quality of the ana-
length dependent and, in the case lytical results.
of stray light, standard dependent
as well. It is not practicable to per- It should be noted that compli-
form all tests at all wavelengths so ance with the above criteria deter-
in practice a few wavelengths, rep- mined with an appropriate set of
resentative of the intended pur- reference standards does not nec-
pose, should be used. The results essarily guarantee that a particular
of these test should be compared analysis can be performed with a
to the instrument performance required accuracy and linearity.
specifications and/or to the instru- Since many parameters are sam-
mental performance requirements ple dependent this can only be
for the methods in use. demonstrated with an appropriate
Performance verification then system suitability test that uses
effectively demonstrates whether the sample itself.
Practical aspects
Whilst liquid standards have the
advantage that an appropriate set
can fulfill the regulatory require-
ments as outlined above they do
have disadvantages when com-
pared to solid standards. Some
attempts have been made to
reduce these disadvantages by
sealing liquid standards into
cuvettes but the results are not
entirely satisfactory. The relative
advantages and disadvantages of
the different types of standards
are compared in table 5.
A practical solution:
liquid standards in ampules
The problems associated with the
preparation and use of liquid stan-
dards have been virtually eliminat-
ed by making them available as
prepared solutions in sealed
ampules. To use them it is only
necessary to break open the
ampule, pour in the curette and
measure. The contents of the stan-
dards kit and the purpose of each
standard are summarized in
table 6.
Solid Standards Liquid Standards
Handling Straightforward, but user Normally prepared by user Straightforward, but user
must take care to avoid using pure chemicals,
dust or scratching. calibrated balance and
volumetric flasks.
User requires some skill to
eliminate cross contamina-
tion. Using liquid standards
is to some extent a test of
proper sample handling.
Calibration User must calibrate each If prepared accurately,
standard individually. these are absolute
standards that do not
require calibration.
Stability Good, but require recalib- If not properly stored,
ration at 1–2 years. some standards show
significant instability.
Standards are best pre-
pared fresh for each test.
Cost High, because each stan- Can be low, because
dard must be individually chemical costs are
calibrated and then recali- minimal and no calibra-
brated at intervals. tion is required. Can also
be prepared in bulk.
Table 5
Comparison of practical aspects of different standard types
Standard Blank
40 g/l holmium oxide in 10 % perchloric acid
10 % perchloric acid
60 mg/l potassium dichromate in 0.01 N sulfuric acid
0.01 N sulfuric acid
50 g/l sodium nitrite in water Water
10 g/l sodium iodide in water Water
12 g/l potassium chloride in water Water
0.02 % v/v toluene in hexane Hexane
Table 6
Contents of liquid standards kit from Agilent Technologies
Comment
must take care to avoid
dust or scratching.
Because the cuvette con-
taining the sample is also a
factor, each standard must
be calibrated individually.
Generally good, but some
(potassium dichromate)
are less stable than solid
standards. User may need
to recalibrate at 6-monthly
intervals.
High, because each standard
must be individually calibra-
ted and then recalibrated at
intervals.
Test
Wavelength accuracy
Photometric accuracy
Stray light at 340 nm
Stray light at 220 nm
Stray light at 200 nm
Resolution
The only additional requirement is
water as blank for the non-critical
stray-light tests, all other refer-
ence solvents are included. The
advantages of these standards are
that they:
• save time because no prepara-
tion is required
• eliminate false OQ/PV failures
due to errors in preparation
• are inexpensive compared to
other calibration standards
• include a certificate of analysis
for traceability, and
• have been shown to have
excellent stability.
Errors due to poor sample han-
dling can still occur but, if desired,
these can be significantly reduced
by using a flow cell instead of a
standard cuvette. The test solu-
tions are drawn into the cell using
a syringe and the potential for
cross contamination is greatly
reduced. A further advantage of
this procedure is that it saves con-
siderable time compared to using
a standard cuvette which must be
rinsed carefully several times
between standards. Test have
shown that a full OQ/PV including
noise and baseline flatness tests
can be performed in about 30 min-
utes on the Agilent 8453 UV-visible
spectrophotometer and its perfor-
mance verification software.
Because diode-array spectropho-
tometers such as the Agilent 8453
have excellent stability and relia-
bility it is only necessary to repeat
the OQ/PV tests at 6–12-month
intervals.
Additional test recommenda-
tions
To ensure continued proper opera-
tion between OQ/PV testing, and
to ensure suitability of the system
for the specific analyses being per-
formed, the following additional
tests are recommended.
Instrument selftest
The Agilent ChemStation software
for UV-visible spectroscopy
includes a set of self-test routines
that check proper electronic and
optical operation of the spec-
trophotometer as well as wave-
length accuracy checks with the
two lines at 486.1 and 656.0 nm
from the deuterium lamp.
System suitability
System suitability should not be
confused with method validation.
System suitability is designed to
evaluate the components of the
analytical system to show that the
performance of the system meets
the standards required by the
method. While method validation
is performed once at the end of
method development, system suit-
ability tests are performed on a
given system periodically to deter-
mine its adequacy or effective-
ness. System suitability require-
ments for chromatography sys-
tems have been well defined but
no similar definition exists yet for
UV-visible spectroscopy systems.
In practice users have developed
their own strategies for perform-
ing system suitability. For exam-
ple:
• Measure and calibrate using
one standard with a concentra-
tion equal to 100 percent of the
expected component concen-
tration. Then measure and
quantify the standard and the
standard diluted by a factor
of 2. The results of both sam-
ples should be within a speci-
fied percentage of the known
concentration. Remeasuring
the standard demonstrates the
quality of the initial measure-
ment.
• Measure the standard and then
measure a series of dilutions of
the standard and calculate the
extinction coefficient
(absorbance/concentration)
for each concentration. The
values of the extinction coeffi-
cients should not vary by more
than a specified percentage.
Summary
Performing OQ/PV that exactly
meets international regulatory
requirements and GLP has, until
now, been very time consuming
and prone to errors. Using liquid
standards in ampules virtually
eliminates these problems.
References 4
National Institute of Standards
1 and Technology, Standard
Alford, J.S., F.L. Cline, F.L., Reference Materials Catalog,
“PMA’s Computer System Section 204, Optical Properties,
Validation Committee, Computer pages 104–106, 1995,
System Validation—Staying Gaithersburg, MD, USA
Current,: Installation
Qualification,” Pharmaceutical 5
Technology, 1990, September, European Pharmacopoeia, 3rd
88–104 Edition, Section 2.2.25,
“Absorption Spectrophotometry,
2 Ultraviolet and Visible,” pages
“EURACHEM Guidance Document 28–29, 1996, Strasbourg, France
No. 1/WELAC Guidance
Document No. WGD 2:
Accreditation for chemical labora-
tories: Guidance on the interpreta-
tion of the EN 45000 series of
standards and ISO/IEC Guide 25,”
1993
3
United States Pharmacopeia
XXIII/National Formulary XVIII,
“Spectrophotometry and light-
scattering,” General Chapter 851,
page 1830, 1996, The United
States Pharmacopoeia
Convention, Inc., Rockville, MD,
USA
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